Post-transcriptional regulation of messenger RNA (mRNA) stability and translation are important control points for gene expression. The overall goal of this project is to generate and utilize structural information to enhance our understanding of these processes. We are conducting structural and biochemical studies of proteins that are involved in or affect the mechanism of RNA silencing. RNA silencing, the destruction of mRNA by double stranded RNA containing corresponding sequences, has proven to be a useful tool to knock out expression of target genes in eukaryotic cells and may have therapeutic potential. In the past few years, much has been learned about the mechanism by which RNA silencing occurs, including the identification of proteins involved in the process. We are studying the structure of a plant viral protein that suppresses RNAi by binding to the intermediate RNAs, small interferring RNAs (siRNAs), that direct mRNA destruction. We have determined the crystal structure of the p19 protein from Carnation Italian Ringspot Virus in complex with an siRNA. Biochemical studies have established important factors for protein:RNA recognition. Additional biochemical studies are ongoing to examine the ability of p19 protein to inhibit microRNA directed processes. We are also studying other proteins that suppress RNA silencing and are involved in the RNA silencing pathway. We are using structural and biochemical methods to understand their functions. In addition we study Pumilio family proteins. We have determined the crystal structure of human Pumilio 1 protein alone and in complex with RNA. These structures have provided rules for sequence-specific RNA recognition by this family of proteins. We are using this information to create RNA-binding proteins with altered RNA sequence specificity.